Release of chemical transmitters by regulated exocytosis underlies many forms of intercellular communication, including hormone release and synaptic transmission. Exocytosis is subject to complex modulation and involves a web of protein-protein interactions and membrane remodeling events. G protein-coupled receptors (GPCRs) play a central role in orchestrating this complex regulation, and Gi/o- coupled GPCRs are well known to inhibit transmitter release from neurosecretory cells by release of G protein ?? subunits. This profound inhibition has the potential to contribute to presynaptic integration and synaptic plasticity. The best-studied mechanism for this inhibition is modulation of the voltage sensitivity of Ca2+ channels. However, G?? can also directly inhibit neurotransmitter release at a point distal to Ca2+ entry by binding to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins as well as the assembled SNARE complex. We have shown that the C-terminus of SNAP25 is critical for the ability to inhibit transmitter release. We have devised an assay of G??-SNAP25 interaction using the AlphaScreen, and have screened a small library of compounds that were designed based on known chemotypes which modulate protein-protein interaction (PPI) for both inhibitors and enhancers of this. In this grant, we will optimize both inhibitors and enhancers of G??-SNAP25 interaction In Aim 1, we will carry out medicinal chemical optimization of both classes of PPI compounds to increase their potency, selectivity, and bioavailability. In Aim 2, we will determine the selectivity of the compounds for G??-SNAP25 interaction compared to other G??-interacting proteins. In Aim 3, we will determine the effects of the optimized molecules on neurotransmitter release from hippocampal neurons in culture, and determine whether they synergize with the agonists and antagonists of presynaptic Gi/o-coupled GPCRs. These compounds should allow us to evaluate the importance of G??-SNARE interaction for GPCR modulation of exocytosis. They may work in parallel with agonists or antagonists of presynaptic GPCRs, and thus synergize with presynaptic functions, selectively affecting presynaptic but not postsynaptic actions of neurotransmitters. The investigations we propose address a fundamental problem in neuroscience, the molecular mechanisms by which neurotransmitters control exocytosis. These studies may define targets for the development of new therapies that may shed light on the pathological basis of diseases related to secretion and neuromodulation.